The principal pharmacological use of streptokinase is in the promotion of clot lysis. Streptokinase (SK), a 47-kDa protein overproduced by .beta.-hemolytic Streptococci Groups A, B, and C forms a 1:1 stoichiometric complex with human plasminogen (Pg). While no proteolytic activity has been associated with streptokinase, in vivo formation of the heterogeneous SK-Pg dimer (activator complex) results in conformational changes to the plasminogen moiety leading to exposure of an enzymatic active center. This center is capable of proteolytic cleavage of uncomplexed zymogenic plasminogen into the fibrinolytic enzyme Plasmin (Pn). Plasmin cleaves the insoluble protein polymer fibrin, a major structural component of the thrombus, into small, soluble degradation products. Sufficient degradation of fibrin elements results in clot dissolution and lysis.
Thrombus formation is characterized by rapid conformational changes to blood platelets and activation of various plasma proproteins. In response to a range of triggering stimuli, zymogenic prothrombin is catalyzed to thrombin. In turn, thrombin acts upon the soluble structural protein fibrinogen, cleaving the N-terminal A and B polypeptides from the .alpha. and .beta. chains to form fibrin monomer. Cleavage results in redistribution of charge density and exposure of two polymerization sites, enabling growth of the monomer into an insoluble, three dimensional polymeric network. Concurrently, thrombin, in the presence of ADP and the divalent cation Ca.sup.2+, acts to induce significant physiological changes to a "resting" or inactive blood platelet, including thromboxane A.sub.2 synthesis and the release of ADP from intraplatelet storage granules. Such activated platelets are capable of binding fibrinogen, polymerizing fibrin monomer, and fibrin polymer at the platelet GPIIB/IIIA glycoprotein. This binding results in the rapid formation of a three dimensional hemostatic plug, which serves to rapidly induce loss of circulatory system integrity.
Thrombus formation in the absence of vessel trauma or rupture is pathogenic, and is a causative factor in ischemic heart disease (myocardial infarction, unstable angina), ischemic stroke, deep vein thrombosis (DVT), pulmonary embolism (PE), and related conditions.
Appearance of atherosclerotic plaques within the coronary arteries is the precursor to ischemic heart disease (IHD). Disruption of the endothelial layer of coronary arteries by lipid-filled foam cells is followed by microlesions in or rupture of the endothelial wall. Either Event results in exposure of platelet activation compounds within the intima, including tissue factor plasminogen activator and collagen. Platelet aggregation results in thrombus formation at the site of plaque rupture. Mural thrombi extend within this ruptured plaque into the vessel volume. Small, non-occlusive mural thrombi may oscillate in response to pressure variations within the vessel, resulting in transient stenosis of the affected channel. Such time-variant blockage is characteristic of unstable angina. Larger, occlusive mural thrombi may completely block the affected vessel, resulting in myocardial infraction and/or patient death.
Causative factors for ischemic stroke include cardiogenic emboli, atherosclerotic emboli, and penetrating artery disease. Cardiogenic emboli are generated within the left atrium and ventricle as a result of valve disease or cardiomyopathy. Migration of the embolus through the aorta into the carotids results in stenosis of a cerebral vessel. As in IHD, atherosclerotic plaques within the carotids or cerebral vasculature serve as loci for the formation of mural thrombi. Vascular disease can result in hypercoagulative states, resulting in thrombus formation. Consequences of ischemic stroke include loss of function of the affected region and death.
Pulmonary embolism results from the migration of the embolus from a formation site within the deep veins of the extremities into the pulmonary vasculature. In the event of an acute blockage, consequences include rapid death by heart failure. Pulmonary hypertension frequently results.
Formation of emboli within the deep veins of the lower extremities is characterized as deep vein thrombosis. Causative factors include atherosclerotic plaques and blood stasis. Certain surgical procedures also correlate strongly with postoperative venous clot formation. These include hip or knee replacement, elective neurosurgery, and acute spinal cord injury repair.
Thrombolytic Therapy
Therapeutic lysis of pathogenic thrombi is achieved through administration of thrombolytic agents. Benefits of thrombolytic therapy include rapid lysis of the thromboembolic disorder and restoration of normal circulatory function. Complications include internal and external bleeding due to lysis of physiologic clots, and stroke resulting cerebral hemorrhage. Currently available treatments are presented in tabular format below. Each of these agents promotes the activation of the proenzyme plasminogen into the fibrin degrading protease plasmin.
______________________________________ Characteristic Streptokinase Anistreplase Urokinase tPA ______________________________________ Molecular 47 131 31-55 70 weight (kDa) Plasma Clear- 15-25 50-90 15-20 4-8 ance time (min) Fibrin Minimal Minimal Moderate Moderate Specificity Plasminogen Indirect Indirect Direct Direct Binding Potential Yes Yes No No Allergic Reaction Approximate $200-300 $2000 $2750 $2200 Cost Typical Dose 1.5 million 30 units 2 million 15 mg units units Administration 1 hr IV 5 min IV 1 million 15 mg infusion infusion unit IV bolus, bolus, then 1 million 0.75 mg/ units IV kg over over 1 hr 30 min ______________________________________
The efficacy of thrombolytic therapy in the treatment of myocardial infarction was demonstrated by the APSAC intervention mortality study. Patients presenting within six hours of onset of acute myocardial infarction (AMI) received intravenous APSAC or a placebo. The APSAC group demonstrated a 47% reduction in mortality over the control. Studies assessing the relative merits of competing thrombolytics in the treatment of acute myocardial infarction include GISSI-2, ISIS-3, GUSTO. GISSI-2 compared tissue plasminogen activator (tPA) vs. streptokinase administered within six hours of acute myocardial infarction. Overall mortality was similar (tPA 9.0%, SK, 8.6%). Reinfarction was significantly lower with tPA (tPA 1.9%, SK 2.3%). Incidence of hemorrhagic stroke was similar (tPA 0.3%, streptokinase 0.25%), while major bleeds were higher with SK (tPA 0.5%, streptokinase 1.0%).
A subsequent meta-study encompassing GISSI-2 and 8401 additional patients resulted in similar results; overall mortality: tPA 8.9%, SK 8.5%; hemorrhagic stroke: tPA 0.6% SK 0.4%; major bleeds: tPA 0.7%, SK 0.8%. I,3IS-3 compared SK vs. tPA vs. anistreplase in patients presenting within 24 hr of AMI. Overall mortality rates were comparable (SK 10.6%, tPA 10.3%, APSAC 10.5%). Major bleeds were somewhat more frequent with APSAC and SK (SK 0.9%, tPA 0.8%, APSAC 0.1%). Significantly fewer instances of hemorrhagic stroke were observed with SK (SK 0.2%, tPA 0.7%, APSAC 0.5%). Reinfarction results were similar to GISSI-2 (SK 3.5%, tPA 2.9%, ASPAC 3.6%). tPA was thus noted to provide more rapid coronary artery patency than SK or APSAC. GUSTO mortality results were again, comparable, with the advantage to tPA (SK 7.3%, tPA 6.3%).
Establishment of patency within the occluded artery varies with time and thrombolytic administered. An accelerated tPA regimen resulted in highest patency rates at 90 minutes (accelerated tPA 83%, tPA 70%, APSAC 70%, UK 60%, SK 54%). However, patency rates converge rapidly over the next 90 minutes and are substantially equivalent at 24 hours.
Successful application of thrombolytics in ischemic stroke has not been realized. Primary factors mitigating against EQU X.sub.1 X.sub.2 X.sub.3 X.sub.4 -Gly-Asp-X.sub.5 X.sub.6 X.sub.7 X.sub.8(SEQ ID NO: 1)
wherein
X.sub.1 is zero or at least one amino acid. If X.sub.1 is one amino acid, it is either the positively charged residue Lys or Arg; PA1 X.sub.2 is Cys or an amino acid analog capable of forming a bridge; PA1 X.sub.3 is zero or at least one amino acid; PA1 X.sub.4 is the positively charged residue Lys or Arg; PA1 X.sub.5 is selected from among Ala, Val, Phe, Pro, Met, Ile, Leu, or Trp; PA1 X.sub.6 is absent or is Pro or Gly; PA1 X.sub.7 is Cys or an amino acid analog capable of forming a bridge; PA1 X.sub.8 is zero or at least one amino acid. If X.sub.8 is one amino acid, it is either the positively charged residue Lys or Arg; PA1 a DNA sequence encoding the first part of a fusion protein, said DNA sequence encoding streptokinase. In a preferred embodiment, the streptokinase is derived from Streptococcus equisimilis strain H46A; PA1 a polylinker or restriction sequence; PA1 a DNA sequence encoding the peptide X.sub.1 X.sub.2 X.sub.3 X.sub.4 -Gly-Asp-X.sub.5 X.sub.6 X.sub.7 X.sub.8 wherein PA1 X.sub.1 is absent or at least one of the group consisting of Lys and Arg; PA1 X.sub.2 is Cys or an amino acid analog capable of forming a bridge; PA1 X.sub.3 is absent or at least one amino acid; PA1 X.sub.4 is the positively charged residue Lys or Arg; PA1 X.sub.5 is selected from the group consisting of Ala, Val, Phe, Pro, Met, Ile, Leu, and Trp; PA1 X.sub.7 is Cys or an amino acid analog capable of forming a bridge. PA1 X.sub.8 is zero or at least one amino acid. PA1 a DNA sequence encoding a peptide X.sub.1 X.sub.2 X.sub.3 X.sub.4 -Gly-Asp-X.sub.5 X.sub.6 X.sub.7 X.sub.8 (SEQ ID NO:1) according to claim 1; PA1 a polylinker or restriction sequence; PA1 a DNA sequence encoding the first part of a fusion protein, said DNA sequence encoding streptokinase; PA1 a polylinker or restriction sequence; and PA1 a DNA sequence encoding the peptide X.sub.1 X.sub.2 X.sub.3 X.sub.4 -Gly-Asp-X.sub.5 X.sub.6 X.sub.7 X.sub.8 (SEQ ID NO:1). PA1 1. Preferential ligation of streptokinase derivatives to the acute thrombus resulting in: PA1 X.sub.1 is zero or at least one amino acid. If X.sub.1 is one amino acid, it is either the positively charged residue Lys or Arg; PA1 X.sub.2 is Cys or an amino acid analog capable of forming a bridge; PA1 X.sub.3 is zero or at least one amino acid; PA1 X.sub.4 is the positively charged residue Lys or Arg; PA1 X.sub.5 is selected from among Ala, Val, Phe, Pro, Met, Ile, Leu, or Trp; PA1 X.sub.6 is absent or is Pro or Gly; PA1 X.sub.7 is Cys or an amino acid analog capable of forming a bridge; PA1 X.sub.8 is zero or at least one amino acid. If X.sub.8 is one amino acid, it is either the positively charged residue Lys or Arg; PA1 a DNA sequence encoding the first part of a fusion protein, said DNA sequence encoding streptokinase. In a preferred embodiment, the streptokinase is derived from Streptococcus equisimilis strain H46A; PA1 a polylinker or restriction sequence; PA1 a DNA sequence encoding the peptide X.sub.1 X.sub.2 X.sub.3 X.sub.4 Gly-AspX.sub.5 X.sub.6 X.sub.7 X.sub.8 (SEQ ID NO:1) wherein PA1 X.sub.1 is absent or at least one of the group consisting of Lys and Arg; PA1 X.sub.2 is Cys or an amino acid analog capable of forming a bridge; PA1 X.sub.3 is absent or at least one amino acid; PA1 X.sub.4 is the positively charged residue Lys or Arg; PA1 X.sub.5 is selected from the group consisting of Ala, Val, Phe, Pro, Met, Ile, Leu, and Trp; PA1 X.sub.6 is absent or Pro or Gly; PA1 X.sub.7 is Cys or an amino acid analog capable of forming a bridge. PA1 X.sub.8 is zero or at least one amino acid. PA1 a DNA sequence encoding a peptide X.sub.1 X.sub.2 X.sub.3 X.sub.4 Gly-AspX.sub.5 X.sub.6 X.sub.7 X.sub.8 (SEQ ID NO:1) according to claim 1; PA1 a polylinker or restriction sequence; PA1 a DNA sequence encoding the first part of a fusion protein, said DNA sequence encoding streptokinase; PA1 a polylinker or restriction sequence; and PA1 a DNA sequence encoding the peptide X.sub.1 X.sub.2 X.sub.3 X.sub.4 GDX.sub.5 X.sub.6 X.sub.7 X.sub.8 (SEQ ID NO:1). PA1 1. Preferential ligation of streptokinase derivatives to the acute thrombus resulting in: PA1 2. Relatively high plasminogen to plasmin conversion activity at the surface of the acute throb)us, resulting in: PA1 3. Competitive inhibition between platelet aggregation processes and streptokinase derivatives ligation resulting in: PA1 4. Significant enhancement in potency of low-cost streptokinase, resulting in creation of potentially lowest cost per dosage thrombolytic agent in existence. This reduction in cost can be expected to make thrombolytic therapy more attractive to clinicians, especially within a managed care environment. PA1 5. The combination of low therapeutic dosages, diminished risk of uncontrolled bleeding and low cost may allow the use of streptokinase derivatives of the present invention by emergency or outpatient practitioners as a new modality for immediate therapeutic treatment of time-critical thromboembolic disorders, including ischemic stroke, myocardial infarction, or massive pulmonary embolism.
Specific examples of polypeptides according to the present invention include the following:
The recombinant streptokinase derivatives of the present invention display increased specificity for activated platelets, and thus achieve the above cited design goals.
The streptokinase derivatives of the present invention are produced using molecular cloning techniques in which competent E. coli are transformed with pThioHis expression plasmids. These plasmids carry ligated DNA inserts coding for fusion proteins within the open reading frame of thioredoxin:
A recombinant DNA molecule comprising the following elements in the 5' to 3' direction, wherein said elements are operably linked:
X.sub.6 is absent or Pro or Gly;
Of particular importance are recombinant DNA molecules wherein the peptide is one of the following: EQU Arg-Cys-Gly-Arg-Gly-Asp-Trp-Pro-Cys-Arg (SEQ ID NO:2) EQU Lys-Cys-Gly-Arg-Gly-Asp-Trp-Pro-Cys-Arg (SEQ ID NO:3) EQU Lys-Cys-Gly-Lys-Gly-Asp-Trp-Pro-Cys-Arg (SEQ ID NO:4) EQU Arg-Cys-Gly-Lys-Asp-Gly-Trp-Pro-Cys-Arg (SEQ ID NO:5)
Additionally, the invention comprises a recombinant DNA molecule comprising the following elements in the 5' to 3' direction, said elements being operably linked:
The advantages of the streptokinase derivatives of the present invention are as follows:
a. high local concentrations of streptokinase derivatives; PA2 b. reduced concentrations of streptokinase derivatives in global circulatory system resulting in reduced risk of hemorrhaging or uncontrolled bleeding; PA2 a. high local concentrations of streptokinase derivatives; PA2 b. reduced concentrations of streptokinase derivatives in global circulatory system resulting in reduced risk of hemorrhaging or uncontrolled bleeding; PA2 c. reduced dosage required to achieve therapeutic concentrations local to the thrombus. PA2 a. high local concentrations of plasmin; PA2 b. increased fibrinolytic activity at the locus of the thrombus; PA2 c. reduced thrombolysis time, resulting in faster restoration of normal circulatory function. PA2 a. diminished rates of clot aggregation; PA2 b. reduced thrombolysis time; PA2 c. reduction or elimination of need for separate anti-coagulant therapy.
to an inducible, expressed carrier protein but upstream of its termination sequence. Successful expression is followed by proteolytic cleavage of the carrier protein. The preferred embodiment is the pThioHis vector, which has proven to be useful for large-scale expression of soluble fusions.
pThioHis contains an altered tray gene coding for a mutant thioredoxin with high affinity for divalent cations. This allows for rapid purification of expressed fusions via metal chelate affinity chromatography (MCAC). Specifically Glu-31, Glu-63 in wild-type thioredoxin have been mutated to His-31, His-63. Tertiary interaction with the His-7 site results in a histidine patch, with the aforementioned affinity for chelate species. 3' to trxA is an enterokinase recognition and cleavage site (Asp-Asp-Asp-Asp-Lys) (SEQ ID NO:11), which provides for cleavage of the desired protein form the thioredoxin carrier following chromatographic purification. 3' to the enterokinase site is a polylinker, or multiple cloning site (MCS) to facilitate oriented ligation of the desired insert. Expression of the fusion is controlled by the inducible trc(trp-lac) promoter. During amplification, expression is halted by the lacI.sup.q repressor, located on the plasmid. Addition of isopropyl-.beta.D-thiogalactopyranoside (IPTG) results in disassociation of the lacI.sup.q repressor product from the lacO operator, allowing transcription.